Background
Germline mutations in
BRCA1 (MIM 113705) and
BRCA2 (MIM 600185) are associated with increased risk of breast and ovarian cancer. The discovery of germline mutations has led to the accurate identification of individuals who are at risk of cancer and the development of new therapies for the disease. In many countries, individuals with
a priori risk of 10–20% of having inherited a germline mutation in
BRCA1 and
BRCA2 are offered genetic counselling and testing [
1] and on average, 17–20% of the genetic testing for
BRCA1 and
BRCA2 detect pathogenic mutations [
2,
3]. However, approximately 10–20% of the tests lead to the identification of variants of uncertain significance (VUS) which comprise missense variants, intronic variants, synonymous variants and in-frame insertions or deletions [
4,
5], for which the clinical relevance remains equivocal.
The frequency of VUS varies by ancestry around the world with lower frequency in populations that are well studied such as the Caucasian population in North America and Europe, and high frequency in populations such as Asian, African and Middle Eastern where there has been little study and limited availability of genetic counselling and testing [
6]. Over time, VUS are systematically reclassified as additional information or evidence is supported [
7,
8]. According to a study reported by Myriad Genetics in United States based on 20 years of experience, the frequency of VUS in
BRCA1 and
BRCA2 declined from 12.8 to 2.1% [
6]. Notably, the frequency of VUS remains the highest in Asians with 7% of the tests reported as VUS. Although the pathogenicity of VUS has been studied using different approaches such as multifactorial likelihood model, population frequency, functional or mRNA splicing assay, the majority of these are focused on VUS in the Caucasian population [
9‐
11]. In Asia, studies have reported novel variants but the clinical significance of these variants has not been further investigated [
12‐
15].
In this study, we analyzed the frequency of 7 BRCA1 and 25 BRCA2 variants in an Asian cohort of 2,110 breast cancer patients and 1,493 healthy women, in which the pathogenicity of variants is evaluated using a case-control approach.
Results
We determined the spectrum of
BRCA1 and
BRCA2 deleterious mutations and variants in 467 breast cancer patients by full sequence analysis and large genomic rearrangement analysis. Of these, 69 (14.8%) had germline deleterious mutations and 125 (26.8%) had variants in
BRCA1 and
BRCA2 genes. In total, 24
BRCA1 and 45
BRCA2 missense and intronic variants were identified from 109 individuals. Of these 69 variants, 67 variants that could be designed for the MassARRAY platform were included in a multiplex genotyping assay (Additional file
1: Table S1a and S1b).
The genotyping assay was tested on 2,110 breast cancer cases and 1,493 healthy controls (Table
1). Majority of the individuals recruited for this study were Chinese (66.1% in cases and 71.7% in controls), followed by Malay (19.2% in cases and 11.2% in controls) and Indian (14.7% in cases and 17.1% in controls). The average age of breast cancer cases (49.5 years) was slightly younger than healthy controls (50.3 years). Notably, there was no difference in age for cases and controls for Chinese and Indian women, but healthy women were on average 2 years older than the cases for Malay women (Additional file
1: Table S2). Ethnicity, age and family history of breast or ovarian cancer were significantly associated with breast cancer risk and hence were included as covariates in single-variant association testing.
Table 1
Characteristics of Malaysian breast cancer cases and healthy controls
Ethnicity | <0.001 |
Chinese | 1,394 | 66.1 | 1,071 | 71.7 | |
Malay | 406 | 19.2 | 167 | 11.2 | |
Indian | 310 | 14.7 | 255 | 17.1 | |
Agea (years) | |
Average age | 49.5 | | 50.3 | | 0.009 |
≤ 30 | 76 | 3.6 | 0 | 0 | |
31–40 | 337 | 16.0 | 72 | 4.8 | |
41–50 | 751 | 35.6 | 781 | 52.3 | |
51–60 | 623 | 29.5 | 501 | 33.6 | |
≥ 61 | 323 | 15.3 | 139 | 9.3 | |
Family historyb
| 0.001 |
Yes | 506 | 24.0 | 288 | 19.3 | |
No | 1,598 | 75.7 | 1,205 | 80.7 | |
No data | 6 | 0.3 | 0 | 0 | |
Pathology profile | |
ER+ | 1,218 | 57.7 | | | |
ER- | 451 | 21.4 | | | |
Triple negative | 242 | 11.5 | | | |
No data | 199 | 9.4 | | | |
Genotyping of 2 BRCA1 (c.-19-3A > G and c.-19-10 T > C) and 2 BRCA2 (p.His523Arg and p.Arg2502His) variants resulted in genotyping call rates of <95%, and these variants were therefore excluded from the analysis. We also excluded 19 cases and 29 controls from analysis because these samples were failed to be genotyped in >20% of the assays. As a result, 2,091 breast cancer patients and 1,464 healthy controls were analyzed using a case-control approach. The genotyping cohort also included 120 individuals who were previously analyzed by germline analysis. Of these, genotyping was concordant with sequencing in 119 of 120 individuals (99.2%). Approximately 5% of randomly selected samples (88 cases and 67 controls) were duplicated in the genotyping assay and the concordance rate among the duplicated samples was 98.7% (153/155).
We identified
BRCA2 p.Ile3412Val with variant frequency >2% in unaffected women and an additional four variants (
BRCA2 p.Cys315Ser, p.Ile1929Val, p.Arg2108Cys and p.Lys2729Asn) with variant frequency >1% in unaffected women (Table
3). All of these are unlikely to be associated with increased risk of breast cancer.
Of the 63 variants that were included in the analysis, thirty-one variants (14
BRCA1 and 17
BRCA2) could not be evaluated as carriers were present only in either breast cancer cases or healthy controls. Only 32 variants (7
BRCA1 and 25
BRCA2) were present in both cases and controls and these were analyzed for association with breast cancer risk (Tables
2 and
3).
Table 2
BRCA1 variants detected in Malaysian carriers by genotyping
c.571G > A | p.Val191Ile | 10 | C0 | Benign | Damaging | 0.30 | 0.75 | 5/1218 | 11/1464 | 0.53 | 0.18–1.54 | 0.242 |
c.823G > A | p.Gly275Ser | 4 | C0 | Probably damaging | Damaging | 0.08 | 0.14 | 4/2089 | 2/1464 | 1.49 | 0.27–8.25 | 0.650 |
c.1036C > T | p.Pro346Ser | 4 | C0 | Benign | Tolerated | 0.13 | 0.14 | 5/1219 | 2/1464 | 3.30 | 0.64–17.09 | 0.155 |
c.2286A > T | p.Arg762Ser | 3 | C0 | Benign | Damaging | 0.13 | 0.07 | 6/1218 | 1/1463 | 7.44 | 0.89–62.32 | 0.064 |
c.2726A > T | p.Asn909Ile | 2 | C0 | Possibly damaging | Damaging | 0.13 | 0.27 | 5/2091 | 4/1462 | 0.90 | 0.24–3.37 | 0.874 |
c.3625 T > G | p.Leu1209Val | 1 | C0 | Possibly damaging | Damaging | 0.04 | 0.14 | 1/2058 | 2/1461 | 0.18 | 0.02–2.05 | 0.168 |
c.3662A > C | p.Glu1221Ala | 3 | C0 | Probably damaging | Damaging | 0.07 | 0.21 | 1/1215 | 3/1454 | 0.44 | 0.05–4.19 | 0.471 |
Table 3
BRCA2 variants detected in Malaysian carriers by genotyping
c.215A > G | p.Asn72Ser | 1 | C0 | Benign | Tolerated | 0.07 | 0.21 | 1/1215 | 3/1454 | 0.41 | 0.04–3.98 | 0.441 |
c.440A > G | p.Gln147Arg | 16 | C0 | Benign | Tolerated | 0.39 | 0.62 | 12/1218 | 9/1463 | 1.38 | 0.58–3.33 | 0.469 |
c.943 T > A | p.Cys315Ser | 15 | C0 | Benign | Tolerated | 0.50 | 1.20 | 18/2055 | 17/1420 | 0.79 | 0.41–1.55 | 0.496 |
c.1825C > G | p.Gln609Glu | 1 | C0 | Benign | Damaging | 0.06 | 0.07 | 2/1218 | 1/1464 | 1.65 | 0.15–18.37 | 0.683 |
c.2186 T > C | p.Ile729Thr | 0 | C0 | Benign | Tolerated | 0.09 | 0.28 | 1/1213 | 4/1423 | 0.31 | 0.04–2.80 | 0.298 |
c.3445A > G | p.Met1149Val | 8 | C0 | Benign | Tolerated | 0.26 | 0.48 | 7/1215 | 7/1454 | 0.83 | 0.29–2.43 | 0.740 |
c.4376A > G | p.Asn1459Ser | 1 | C0 | Benign | Tolerated | 0.10 | 0.14 | 5/2087 | 2/1462 | 1.89 | 0.37–9.80 | 0.448 |
c.4779A > C | p.Glu1593Asp | 3 | C0 | Benign | Tolerated | 0.14 | 0.27 | 6/2088 | 4/1464 | 1.15 | 0.32–4.13 | 0.834 |
c.5167A > C | p.Thr1723Pro | 1 | C0 | Possibly damaging | Tolerated | 0.04 | 0.07 | 1/1214 | 1/1454 | 1.05 | 0.06–16.99 | 0.976 |
c.5312G > A | p.Gly1771Asp | 44 | C0 | Benign | Tolerated | 0.03 | 0.07 | 1/2054 | 1/1459 | 0.58 | 0.04–9.74 | 0.706 |
c.5785A > G | p.Ile1929Val | 32 | C0 | Benign | Tolerated | 0.82 | 1.62 | 20/1213 | 23/1421 | 1.03 | 0.56–1.90 | 0.916 |
c.5986G > A | p.Ala1996Thr | 4 | C55 | Probably damaging | Tolerated | 0.16 | 0.34 | 6/2060 | 5/1462 | 0.95 | 0.29–3.16 | 0.932 |
c.6322C > T | p.Arg2108Cys | 22 | C0 | Benign | Tolerated | 0.55 | 1.12 | 13/1207 | 16/1431 | 0.94 | 0.45–1.98 | 0.871 |
c.6325G > A | p.Val2109Ile | 8 | C0 | Benign | Tolerated | 0.25 | 0.49 | 6/1213 | 7/1422 | 0.89 | 0.30–2.69 | 0.837 |
c.7052C > G | p.Ala2351Gly | 6 | C0 | Possibly damaging | Damaging | 0.21 | 0.49 | 8/2077 | 7/1422 | 0.78 | 0.28–2.17 | 0.627 |
c.7469 T > C | p.Ile2490Thr | 240 | C0 | Benign | Tolerated | 0.06 | 0.07 | 3/2060 | 1/1462 | 2.27 | 0.24–21.90 | 0.480 |
c.8187G > T | p.Lys2729Asn | 24 | C0 | Probably damaging | Damaging | 0.63 | 1.30 | 15/1218 | 19/1463 | 0.98 | 0.50–1.95 | 0.964 |
c.8356G > A | p.Ala2786Thr | 2 | C0 | Probably damaging | Damaging | 0.17 | 0.28 | 8/2088 | 4/1448 | 1.52 | 0.45–5.07 | 0.498 |
c.8702G > A | p.Gly2901Asp | 3 | C65 | Probably damaging | Damaging | 0.11 | 0.34 | 1/1217 | 5/1464 | 0.25 | 0.03–2.16 | 0.208 |
c.9104A > G | p.Tyr3035Cys | 3 | C55 | Probably damaging | Damaging | 0.06 | 0.07 | 2/1213 | 1/1454 | 2.34 | 0.21–26.02 | 0.489 |
c.9538C > T | p.Leu3180Phe | 1 | C0 | Probably damaging | Damaging | 0.04 | 0.14 | 1/2089 | 2/1448 | 0.34 | 0.03–3.81 | 0.382 |
c.10234A > G | p.Ile3412Val | 114 | C0 | Benign | Tolerated | 1.42 | 2.53 | 40/1217 | 37/1464 | 1.29 | 0.81–2.04 | 0.280 |
c.68-7 T > A | - | 3 | Not applicableh
| 0.07 | 0.07 | 4/2076 | 1/1458 | 3.10 | 0.34–28.31 | 0.316 |
c.516 + 18 T > C | - | 1 | Not applicableh
| 0.29 | 0.49 | 8/1207 | 7/1417 | 1.41 | 0.51–3.91 | 0.511 |
c.8954-5_8954-2delAACA | - | 0 | Not applicableh
| 0.06 | 0.14 | 2/2088 | 2/1463 | 0.83 | 0.12–5.93 | 0.855 |
In the single-variant association testing using logistic regression,
BRCA1 p.Arg762Ser was associated with breast cancer risk with a marginal significance (OR = 7.4; 95% CI, 0.9–62.3;
p = 0.06) (Additional file
2: Figure S1). This variant was found in 5 out of 858 Chinese breast cancer patients (0.6%) and 1 out of 1,054 Chinese controls (0.1%) (Additional file
1: Table S3), and was marginally associated with breast cancer risk in Chinese women (OR = 6.7; 95% CI, 0.8–57.6;
p = 0.08) (Additional file
2: Figure S2). The average age of diagnosis was 39 years old, 25% of women had estrogen receptor (ER) negative breast cancer but none reported any family history of breast or ovarian cancer. The
BRCA1 p.Pro346Ser was associated with breast cancer risk in Chinese women [5 out of 859 Chinese breast cancer patients (0.6%) and 2 out of 1,055 Chinese controls (0.2%), OR = 3.3; 95% CI, 0.6–17.3;
p = 0.15] (Additional file
1: Table S3a and Additional file
2: Figure S2), but the results were not statistically significant. The average age of diagnosis was 62 years old, 20% of women had ER negative breast cancer but none reported any family history of breast or ovarian cancer. None of the
BRCA2 variants were significantly associated with breast cancer risk either in the overall cohort, or when stratified by ethnicity (Additional file
2: Figure S1 and S2).
The probability that missense variants were deleterious to protein function was assessed by three
in-silico models, namely AGVGD, PolyPhen-2 and SIFT. Of the 29 missense variants that have been analyzed, three (
BRCA2 p.Ala1996Thr, p.Gly2901Asp and p.Tyr3035Cys) were predicted to be likely pathogenic (Tables
2 and
3).
Discussion
In this study, we analyzed the frequency of 7
BRCA1 and 25
BRCA2 variants from exonic and intronic regions identified previously in Malaysian breast cancer patients by germline analysis. The genotyping of variants was conducted using a high-throughput mass spectrometry platform [
18].
The variant frequency suggested that one of the 63 tested variants has a minor allelic frequency of >2% in unaffected women and is likely to be benign [
6]. Four variants that had more than 1% of variant frequency in unaffected women could be potentially benign. Of these variants, three (
BRCA2 p.Ile1929Val, p.Lys2729Asn and p.Ile3412Val) have been classified as Class 1 (not pathogenic) and one (
BRCA2 p.Arg2108Cys) has been classified as Class 2 (likely not pathogenic) in either Breast Cancer Information Core Database (
http://research.nhgri.nih.gov/bic/) or DatabaseARUP Laboratories
BRCA Mutation Database (
http://arup.utah.edu/database/BRCA/) using different approaches [
20,
23]. Moreover,
BRCA2 p.Ile3412Val was also reported by ENIGMA (
http://enigmaconsortium.org/) as benign variant to occur in non-founder African control reference group at an allele frequency ≥1%. Although
BRCA2 p.Lys2729Asn was predicted to have damaging effect by PolyPhen-2 and SIFT, the prediction models may have limitation to predict the actual consequences of missense mutation accurately [
24] therefore the population frequency supersedes the prediction models [
8]. These findings are in accordance with the results of our study which concluded that these variants are benign variants or polymorphisms.
The clinical significance of
BRCA2 p.Cys315Ser and p.Arg2108Cys is currently listed as uncertain, but our study suggests that these variants are likely to be benign. This is consistent with a study in Chinese women from Shanghai in which
BRCA2 p.Cys315Ser was detected in 1.4% of cases and 0.9% of controls, compared with 0.9% of cases and 1.2% of controls in our study [
12]. Notably,
BRCA2 p.Arg2108Cys was evaluated as pathogenic in spontaneous homologous recombination [
25], but our study suggests that this variant is unlikely to be associated with high risk of breast cancer.
It was estimated that the rare variant had a relative risk of above 2 and above 4 might confer moderate and high risk of breast cancer, respectively [
26,
27]. Our study suggests that one
BRCA1 variant may be associated with increased risk of breast cancer. The
BRCA1 p.Arg762Ser may be associated with breast cancer risk with a marginal significance (
p = 0.06). This variant was previously found in Chinese and Malay women and the clinical significance is currently unknown [
12,
28,
29]. Although the
in-silico analyses predicted the amino acid substitution of this variant is unlikely to have damaging effect to protein function, our study suggests that this warrants further analyses in Asian women.
Notably, three
BRCA2 variants (p.Ala1996Thr, p.Gly2901Asp and p.Tyr3035Cys) which are predicted to be pathogenic by
in-silico prediction models were found in cases and in healthy controls. The clinical significance of
BRCA2 p.Ala1996Thr is currently listed as uncertain and the substitution of valine at the same codon (p.Ala1996Val) in a Western European woman is also uncertain [
7].
BRCA2 p.Gly2901Asp was suggested as neutral in mouse embryonic stem cell-based functional assay [
30], and predicted to be uncertain in protein likelihood ratios [
31] and homology-directed repair activity [
32]. Although
BRCA2 p.Tyr3035Cys was predicted to be likely deleterious in protein likelihood ratios [
31], this variant did not show any significant association with breast cancer risk in our study.
There are several limitations to this study. The breast cancer cases were not age- and ethnicity-matched with controls in this study, but these variables were adjusted for all variant analyses. Another limitation is that majority of the variants selected for this study are rare in our population. These rare variants were detected in very low frequency, thus decrease the statistical power in a case-control study. Analyses in larger groups are necessary to confirm these findings.
Acknowledgements
We thank the participants for taking part in this study; all staff at the Breast Care Centre of University Malaya Medical Centre and Subang Jaya Medical Centre for the recruitment of patients and controls in this study; SY Yoon, SY Lee, D Lee and MK Thong for the genetic counselling of patients; H Hasmad, N Hassan and K Sivanandan for the assistance with sample processing; and R Moser and D Irwin for assistance with genotyping assay design.